Many disease conditions (such as cancer and heart disease) are characterized by the co-expression of two or more disease marker in a diseased cell or a cell developing the disease. Such changes may be difficult to elucidate. For example, Dewey et al., Circ Cardiovasc Genet. 4:26-35, 2011 describes fifty fetal gene coexpression modules in developing myocardium that were not present in normal adult tissue. Of these fifty, three (6%) were reproduced in hypertrophied myocardium and seven (14%) were reproduced in failing myocardium. One fetal module was common to both failing and hypertrophied myocardium.
Similarly, many cancers show co-expression of disease markers. For example, breast cancer cells co-express several hormonal markers (see, e.g., Yang et al., Cancer Res. 67:10608-17, 2007). Likewise, Hodgkin's disease cells co-express CD20 and CD15 (see Zukerberg et al., Am. J. Pathol. 139: 475-483, 1991).
Methods to detect co-expression of two different markers have been described. For example, fluorescence (or Förster) resonance energy transfer (FRET) is a distance-dependent physical process by which energy is transferred nonradiatively from an excited molecular fluorophore (the donor) to another fluorophore (the acceptor). The use of FRET has been widely described in biology (see, e.g., Didenko, V. V., Biotechniques 31: 1106-21, 2001; Sekar and Periamsary, J Cell Biol. 160: 629-633, 2003; Buntru et al., BMC Biology 2009, 7:81-91, 2009; and Ciruela, F. Curr Opin Biotechnol. 19:338-343, 2008). In fluorescence microscopy, as well as in molecular biology, FRET is a useful tool to quantify co-expression of two markers, as well as molecular dynamics between the two markers, such as protein-protein interactions, protein-DNA interactions, and protein conformational changes. For monitoring the complex formation between two molecules, one of them is labeled with a donor and the other with an acceptor, and these fluorophore-labeled molecules are mixed. When they are dissociated, the donor emission is detected upon the donor excitation. On the other hand, when the donor and acceptor are in proximity (1-10 nm) due to the interaction of the two molecules, the acceptor emission is predominantly observed because of the intermolecular FRET from the donor to the acceptor. For monitoring protein conformational changes, the target protein is labeled with a donor and an acceptor at two loci. When a twist or bend of the protein brings the change in the distance or relative orientation of the donor and acceptor, FRET change is observed. If a molecular interaction or a protein conformational change is dependent on ligand binding, this FRET technique is applicable to fluorescent indicators for the ligand detection. Two typical FRET pairs are cyan fluorescent protein and yellow fluorescent protein. These green fluorescent protein variants are easily attached to host proteins. Resonance energy transfer using bioluminescent molecules (e.g., luciferase) instead of cyan fluorescent protein is called BRET and has also been described.
Additional known methods for specific targeting two or more molecular targets in a biological sample that uses a combinatorial target approach are the bimolecular fluorescence complementation (BiFC) and the proximity ligation assay (PLA) (reviewed in Weibrecht, I., Expert Rev Proteomics. 7:401-409, 2010).
All of the known techniques for detecting more than one target simultaneously in a biological sample, however, are difficult to perform and require expensive reagents and the use of highly specific microscopes and other detection devices. Thus, it would be useful to have a less expensive, easier to perform method to detect co-expression of two or more markers in a biological sample.